1) Nerve-derived vascular branching patterning signals We previously observed that arterial vessels and peripheral nerves (PNs) develop alongside each other in the limb skin. This co-patterning is developed by PN-mediated signal(s) that instructively guide the arterial branching network. Therefore, the limb skin vasculature affords an attractive system in which to study the nature of neuronal signals that control vascular network formation. Using the tissue-specific knockout technology, we have begun to dissect out the PN-derived signals that participate in integrating both branching networks. We showed that PN-derived vascular endothelial growth factor (VEGF)-A functions to control arteriogenesis-arterial differentiation and smooth muscle cell association. We have recently discovered that PN-derived C-X-C motif chemokine ligand (CXCL) 12 controls the nerve-blood vessel alignment (manuscript in preparation). We are now actively working on dissecting the role of CXCR4 receptor signaling activated by CXCL12 in the process of the nerve-vessel alignment in the limb skin. 2) Lymphatic vessel development in the central nervous system Central Nervous System (CNS) has been identified as an immunoprivileged site originally due to the lack of lymphatic vasculature. Although scientists recognize the immunoprivileged microenvironment of the brain and spinal cord, they have yet to explain the mechanism by which the brain and spinal cord prevent lymphangiogenesis but not angiogenesis. Our studies focus on understanding what signals from the CNS block lymphatic vessel development. Although there are no lymphatic vessels in the embryonic spinal cord, blood vessels do exist. We have found that supernatant from cultured embryonic spinal cord prevents the differentiation of ECs into lymphatic ECs, as well as the migration of primary lymphatic but not blood ECs. Now we are extensively working on what factors in the spinal cord supernatant prevent the differentiation and migration of the lymphatic vasculature in the CNS. 3) Interaction of developing coronary vasculature with cardiac neurons An improved understanding of coronary vasculature development could lead to key advancements in treating or correcting ischemic heart disease and myocardial infarction as well as congenital heart diseases. We have postulated that in the embryonic heart, cardiac sympathetic axon pathfinding may involve coronary vascular patterning. It turns out that coronary vascular remodeling precedes cardiac innervation and the vascular patterning helps to guide axonal projection (manuscript in preparation). Further studies are needed to determine the vascular-derived signal(s) that cause coronary vessels and cardiac sympathetic axons to interact. 4) Genetic tools to analyze peripheral vascular development Previous studies demonstrate that heart function and normal blood flow are necessary for vascular development. However, current genetic tools for mice fail to uncouple vascular and heart abnormalities. To circumvent this problem, we are searching for promoters and enhancers that are active in endothelial cells but not active in heart endocardial cells. Using a transgenic technology, we are generating a Cre deleter line using the fragment of GATA2 enhancer, which works in peripheral endothelial cells but not heart endocardial cells. Additionally, we are developing a local gene knockdown system in chick embryos using in ovo electroporation delivery of RNAi and see the role of the angiogenic genes in the peripheral vascular development. 5) Vascular niche for adult neurogenesis Stem cells are established in the niche, a unique and specialized microenvironment. Given the importance of the vascular niche for a variety of stem cells, understanding the paracrine signals for stem cell maintenance has become more important. Our challenge is to utilize a systematic multi-faceted approach to identify and validate the vascular niche signals involved in maintenance, self-renewal, proliferation and differentiation of neural stem cells in the the subventricular zone (SVZ) of the lateral ventricle in the adult brain. We have applied the signal sequence trap method by retrovirus-mediated expression screening to highly purified vascular niche cells from the SVZ vasculature. We are now evaluating the physiological relevance of the candidate niche signals in vitro using neurosphere stem cell assay as well as in vivo using viral or electroporation mediated RNAi knock-down approaches.